Surface expression of proteins via covalent linkage with peptidoglycans in Gram-positive bacteria involves unique sorting signals and Sortase-dependent machinery (Mazmanian et al., Science 285:760-763 (1999)). One of the best-studied systems is the emm6 gene of Streptococcus pyogenes that encodes the M6 structural protein (Fischetti et al., 1990. Mol. Microbiol. 4:1603-1605 (1990)). The M6 proteins have a signature cell wall sorting signal, the Leu-Pro-X-Thr-Gly (LPXTG; SEQ ID NO:9) motif, followed by a stretch of hydrophobic amino acids and finally a sequence containing charged residues (KRKEEN; SEQ ID NO:10), which serves as a cell surface retention signal. These cell wall sorting motifs have been identified in other Gram-positive bacteria including Staphlyococcus, Enterococcus, and Listeria, and Lactobacillus (Navarre and Schneewind, Microbio. Mol. Biol. Rev. 63:174-229 (1999)), but not in Lactobacillus species that colonize the human vagina.
The mucosal membranes of all humans are naturally colonized by bacteria (Tannock. Clin. Rev. Allergy Immunol. 22: 231-53 (2002)). Recent scientific evidence has documented the fact that these bacteria interact closely with cells and tissues of the body to regulate natural biological processes. It has become increasingly evident that this mucosal microflora also contributes substantially to numerous diseases affecting cells and tissues of humans.
Generally, domination of the microflora within the vagina and gastrointestinal tract, by lactobacilli and related bacteria, is associated with good health (Redondo-Lopez et al., Rev. Infect. Dis. 12: 856-72 (1990); Tannock. Clin. Rev. Allergy Immunol. 22: 231-53 (2002)). Natural strains of lactobacilli have been administered for many years as “probiotics” for the purpose of maintaining a healthy microflora within these locations and preventing infection. It is well established that these “healthy bacteria” compete with pathogenic organisms, such as bacteria, viruses and fungi to limit the development and progression of pathogen associated diseases. Nevertheless, this microflora is a fragile and dynamic environment with the natural turnover and disruption of the healthy microflora being associated with the establishment of opportunistic infections. Consequently, approaches to maintain, or even enhance, the integrity and natural properties of the microflora, as a means of preventing or treating disease, would be coveted by the biomedical community.
The mucosal microflora contributes to many local diseases affecting mucosal surfaces. For instance, HIV and other sexually transmitted pathogens must bypass the vaginal mucosa. In addition, the etiology of inflammatory bowel diseases, including ulcerative colitis and Crohn's disease may arise from inappropriate interactions between a disrupted mucosal microflora and cells and tissues of the host. A means of modulating the properties of bacteria within the mucosal flora could aid in the prevention or treatment of these diseases, as well as related conditions affecting mucosal surfaces. Targeting biologically active proteins to the cell wall of these and other organisms could help to treat such diseases.
The present invention addresses these and other problems.